Lipids play key roles in brain function and contribute in important ways to pathologies such as drug addiction, schizophrenia and Alzheimer's disease. In preliminary experiments, we utilized a pipette capture method originally devised for single-neuron mRNA analysis to collect individual somata of dentate gyrus (DG) granule cells - the smallest neurons found in the brain - from living hippocampal slices of adult mice. We analyzed lipid extracts of each granule cell by nanoflow liquid chromatography (nano-LC) coupled to high-resolution time-of-flight mass spectrometry (MS). We were able reliably to detect many important lipids involved in membrane structure, energy storage, and cellular signaling. Importantly, we found that physiological stimulation of the lateral perforant path, a fiber tract that provides major excitatory input to DG granule cells, caused rapid and robust changes in the cells' lipid profile. The present application proposes to develop these initials findings into an optimized and validated protocol that can be widely applied to lipidomics analyses of neurons throughout the brain. We have two specific aims: (1) Method optimization. Our initial protocol is highly sensitive, but has three limits that stem from the vanishingly low amount of biomaterial afforded by a single neuron: (a) it covers only a fraction of the lipidome; (b) it allows tandem MS structure confirmation only for the most abundant lipid species; and (c) it provides relative rather than absolute quantification of detected lipids. We will (i) increase te sensitivity of our procedure through systematic modifications of key analytical parameters; (ii) extend the procedure's quantitative reach; and (iii) build reference libraries of lipid MS data for individual neurons, using pools of individually captured granule DG cells. (2) Method validation. Our preliminary work allowed us to identify a substantial number of lipid species in resting DG granule cells, and to detect specific alterations in the cells' lipid profile following physiologicl stimulation. To test the general applicability of the protocol, we will (i) profile the lipidome of pyramidal neurons in the CA1 and CA3 fields of the hippocampus, which are anatomically and functionally different from granule cells; and (ii) determine the impact of various physiological stimuli on the lipidome of DG granule cells and CA1/CA3 pyramidal neurons. Lastly, to define similarities and differences between single-cell and whole tissue preparations, we will compare the lipidomes of individual granule cells and micropunches of DG tissue under control and stimulated conditions. When fully validated and optimized, the present method will provide a flexible and robust new tool to investigate the roles of lipid molecules in identified neurons isolated from live brain tissue, opening exciting new avenues for research on neural lipids and the role of neuronal diversity in health and disease.